The goal of this project is increase tissue rescue from stroke by improving O2 delivery with the use of cell- free hemoglobin (Hb) O2 carriers (HBOC) and agents that simultaneously improve collateral blood flow. An early clinical attempt to use the first generation of crosslinked tetrameric Hb was not successful partially because of unwanted side effects related to scavenging of the vasodilator, nitric oxide (NO), and production of the vasoconstrictor, endothelin. Our previous work demonstrated that polymerization of Hb avoided some of these side effects and rescued tissue from stroke in animals. However, dilation of pial arteries in the distal ischemic territory adjacent to anastomotic vessels was not sustained, perfusion was not improved, and tissue rescue was likely submaximal. Preliminary data indicated that dilation of these arteries could be better sustained during prolonged ischemia by co-administration of an inhibitor of arginase, an enzyme that can constrain endothelial NO production and promote superoxide generation by uncoupled NO synthase, or by co-administration of nitrite, which enables the NO scavenged by Hb to be recycled by virtue of the nitrite reductase activity of Hb. Moreover, Hb decorated with polyethylene glycol (PEG-Hb) may be superior to previously tested polymerized Hb because of its higher oncotic pressure and viscosity, which promotes endothelial shear stress-induced NO production. Indeed, transfusion of PEG-Hb during early transient focal ischemia was found to markedly reduce cortical infarct volume by 83%. Moreover, polynitroxylation of PEG- Hb, which confers antioxidant properties and limits formation of highly reactive ferryl Hb, was found to improve intraischemic dilation of pial arteries and to rescue cortex by 89%. Aim 1 of this proposal is to optimize the transfusion protocol and dose of PEG-Hb and polynitroxylated PEG-Hb and determine if polynitroxylation provides added benefit in a more severe model of transient ischemia and in an embolic clot model. Aim 2 is to better understand the role of arginase activity in the progressive loss of vasodilation in the ischemic border region. This aim will reveal if an arginase inhibitor reduces endothelial oxidative stress and if co-administration with the HBOC that was optimal in Aim 1 provides additional vasodilation, improved blood flow, and tissue rescue. Aim 3 is to determine if co-administration of nitrite with an HBOC also provides additional vasodilation and tissue rescue. Using the most efficacious vasodilating agents, HBOC product, and transfusion protocol seen in Aims 1-3, the therapeutic window of opportunity, long-term outcome and gender effects will be investigated in an embolic clot model of stroke in Aim 4. Therefore, this proposal will capitalize on more recent advances in our knowledge of the properties of HBOC and vascular control mechanisms to optimize O2 delivery and maximize tissue rescue from ischemic stroke. Results from these studies have the potential to be translated into clinical therapy for the acute treatment of stroke, a disease with high mortality and devastating disabilities that represent a large burden on society.